CN103351422B - Method for extracting natural bioactive protein from unpolished rice or/and rice bran - Google Patents
Method for extracting natural bioactive protein from unpolished rice or/and rice bran Download PDFInfo
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Abstract
The invention discloses a method for extracting natural bioactive protein from unpolished rice or/and rice bran. The method comprises the followings: processing unpolished rice or/and rice bran to obtain degreased rice flour; mixing degreased rice flour and soduim chloride water, then extracting through oscillation, and collecting supernatant fluid through first separation, namely total salt soluble protein solution is obtained; adding alcohol into the total soluble protein solution, mixing uniformly, and removing precipitation part through standing and second separation so as to obtain bioactive protein solution; removing alcohol and salinity, and drying through post-processing to the bioactive protein solution, and then obtaining the natural bioactive protein. The method provided by the invention adopts two food-grade raw materials of sodium chloride and alcohol, and can be used for extracting the natural bioactive protein from the unpolished rice or/and rice bran, the cost is low, and zero contamination is achieved; the natural bioactive protein prepared by the invention can be used for preparing health foods, taken as food additives and applied to cosmetics, and has a wide application prospect.
Description
Technical field
The present invention relates to biological activity protein preparation field, be specifically related to a kind of from brown rice or/and extract the method for natural bioactive albumen rice bran.
Background technology
Rice protein is a kind of food protein of high nutritive value, mainly the Amino acid profile of rice protein is reasonable, its utilization ratio and biological value high, and there is hypoallergenic, be uniquely can in order to avoid allergic experiment com gluten protein (Wang Zhangcun etc. Progress of Study on Rice Protein. Chinese grain and oil journal, 2004,19 (2): 11-17).According to Osborne based on deliquescent sorting technique, rice protein can rough segmentation be white protein, sphaeroprotein, prolamine and gluten.Wherein, white protein and sphaeroprotein can dissolve with sodium-chlor the protein ingredient extracted in rice, also referred to as salting-in protein, it is main physiologically active protein, the rice protein physiological function reported comprise hypotensive, hypoglycemic, remove cholesterol, anticancer change etc. (Wang Liangdong etc. Advance of preparation of rice protein. grain processing, 2007,32 (1): 43-47).The research such as Chanput shows, the white protein extracted in rice rice bran, sphaeroprotein and enzymolysis product thereof all have higher anti-oxidant activity, conventional antioxidant vitamin E and BHT(2 of foodstuffs industry is significantly higher than to Oxidation of Fat and Oils inhibition, 6-di-tert-butyl methyl phenol) (Chanput W, Theerakulkait C, NakaiS.Antioxidative properties of partially purified barley hordein, rice bran proteinfractions and their hydrolysates.Journal of Cereal Sience, 2009, 49 (3): 422-428).
Rice aboundresources, security is good, and rice antioxidation polypeptide Application Areas prospect is boundless, such as, can be used as antioxidant addn oil-control oxidation, the deterioration of the color that minimizing Food Oxidation causes and nutritive value.Because anti-oxidant Toplink removes active oxygen radical superfluous in body effectively; Cell protection and mitochondrial normal configuration; prevent snperoxiaized generation; therefore, rice antioxidation polypeptide can be used as disease that functional food ingredient exploitation prevention and therapy free radical brings out as protective foodss such as cardiovascular diseases, cataract, cancers.Rice antioxidation polypeptide also can be used as repairing type functional component and is applied to makeup, is reached the object delayed senility by scavenging free radicals.
The technology of preparing of rice biologically active polypeptides is for the purpose of antioxidation active peptides, as Qian Junqing etc. has invented the technique that rice gluten is extracted in a kind of utilization from rice processing fent, the acidic aqueous solution that have employed sulfuric acid allotment extracts (Qian Junqing etc., a kind of technics of distilling rice protein, patent publication date 2008.01.02, patent publication No. CN101095455A); Zhang Min etc. have invented a kind of manufacturing technology of rice bran antioxidation active peptides, have employed alkaline process and prepare albumen from rice bran, recycling compound protease enzymolysis obtains the rice bran protein peptide (Zhang Min etc. with anti-oxidant activity, a kind of making of rice bran antioxidation active peptides protein peptide, patent publication date 2011.06.01, patent publication No. CN102080118A); Tian Wei etc. have invented a kind of preparation method of oryzenin antioxidant peptide, wherein have employed basic protein enzymolysis process (Tian Wei etc., a kind of preparation method of Rice Bran Antioxidant Peptide, patent publication date 2010.06.23, patent publication No. CN101748182A).
As mentioned above, the major defect of the Technology of existing rice bioactive peptide is: one is that the preparation great majority of rice protein relate to acid, alkaline purification, and consequent waste water may to environment, and technique is clean not; Two is that the acquisition of rice biologically active peptides all adopts enzymolysis step, and cost is high, complex process; Three are prepared active polypeptide is not natural being present in rice, and is through enzymolysis rice gluten and obtains, and complicated components is uncertain.
Summary of the invention
The invention provides a kind of from brown rice or/and extract the method for natural bioactive albumen rice bran, adopt sodium-chlor and ethanol two kinds of food grade materials, can from brown rice or/and extract natural bioactive albumen rice bran, low cost, pollution-free.
From brown rice or/and extract a method for natural bioactive albumen rice bran, comprise the following steps:
1) by brown rice or/and rice bran obtains degreasing ground rice through skimming treatment;
2) the degreasing ground rice in step 1) is mixed with sodium chloride aqueous solution, then through mechanical shaking extraction, the first separated and collected supernatant liquor, be total salting-in protein solution;
3) to step 2) in total salting-in protein solution add ethanol, mix, through leave standstill, second be separated after remove sediment fraction, obtain solutions of biologically active proteins;
4) solutions of biologically active proteins in step 3) is removed ethanol through aftertreatment, obtains natural bioactive albumen except after salinity and drying.
In the present invention, sodium chloride aqueous solution is adopted to extract the total salting-in protein of rice, obtain total salting-in protein solution, adopt ethanol to carry out removing impurity by means of precipitation to the foreign protein except natural bioactive albumen in total salting-in protein solution, thus after aftertreatment, obtain the higher natural bioactive albumen of purity.
In step 1), degreasing ground rice can adopt commercially available prod also can prepare voluntarily, as preferably, described skimming treatment comprises: by brown rice or/and rice bran crosses 30 ~ 80 orders (further preferably 40 ~ 50 orders) sieve after pulverizing obtain powder material, in powder material, add acetone degreasing, after vibration, separation, drying, obtain degreasing ground rice.Through above-mentioned size screening and through the degreasing ground rice that acetone degreasing obtains, be conducive to extracting natural bioactive albumen from degreasing ground rice.Further preferably, the quality of described powder material and the volume ratio of acetone are 1:2 ~ 4g/mL, i.e. the solid-to-liquid ratio 1:2 ~ 4g/mL of powder material and acetone, further preferably, for 1:3g/mL, adopt the acetone of above-mentioned quality, be conducive to the extraction of follow-up natural bioactive albumen.
Step 2) in, sodium chloride aqueous solution is adopted to extract the total salting-in protein of degreasing ground rice, as preferably, the concentration of described sodium chloride aqueous solution is 0.5mol/L ~ 1.5mol/L, protein solubleness in water depends on the water molecules number on protein molecule around ionic group, namely depends on the hydration levels of protein.Adopt the neutral salt solution of different concns can control the hydration levels of rice protein, namely control the solubleness of protein in rice corn.When adding a small amount of salt in water, salt ion and water molecules are on the impact of the polar group of protein molecule, and protein solubleness in water is increased, and this i.e. the molten phenomenon of salt; But salt concn increase to a certain degree time, the activity of water reduces, and the electric charge of protein surface is neutralized in a large number, and hydration shell is destroyed, so protein is assembled and Precipitation mutually, Here it is saltouts.The neutral salt that the present invention selects is sodium-chlor, sodium chloride concentration suitable in the aqueous solution is the key affecting the total salting-in protein extraction yield of rice, the sodium chloride aqueous solution of 0.5mol/L ~ 1.5mol/L concentration is conducive to salting-in protein and extracts, and rice total salting-in protein yield is higher.
The quality (g) of described degreasing ground rice and the volume (mL) of sodium chloride aqueous solution are than being 1:5 ~ 12g/mL, the i.e. solid-to-liquid ratio 1:5 ~ 12g/mL of degreasing ground rice and sodium chloride aqueous solution, solid-to-liquid ratio refers to the ratio of the quality (g) of degreasing ground rice and the volume (mL) of sodium chloride aqueous solution.When solid-liquid is smaller, a small amount of NaCl solution is unfavorable for the abundant dissolving of albumen; Too high solid-to-liquid ratio (higher than 1:12) is unfavorable for follow-up concentrated, dry, and energy consumption is increased.Under solid-to-liquid ratio 1:5 ~ 12g/mL, rice total salting-in protein yield is higher.
As preferably, the condition of described mechanical shaking extraction is: 20 ~ 40 DEG C of mechanical shaking extractions 2 ~ 4 hours, this vibration temperature can improve protein dissolution speed, and extracts 2 ~ 4 hours, makes rice total salting-in protein yield higher.
As preferably, described first condition be separated is: at the centrifugal 10 ~ 30min of 5000 ~ 8000rpm, first object be separated is separated with insoluble coarse rice powder residue by the total salting-in protein extracting solution being dissolved in sodium chloride aqueous solution, centrifugal force and centrifugation time can affect the effect that residue precipitates, centrifugal rotational speed is too low or the time is too short, residue precipitation not exclusively, extracting solution muddiness is caused to be unfavorable for that the later stage is separated, the too high overlong time of centrifugal rotational speed, energy consumption is increased and causes centrifugal heat production to cause protein denaturation even starch pasting, optimal separation effect can be ensured at the centrifugal 10 ~ 30min of 5000 ~ 8000rpm.
In step 3), adopt ethanol to carry out removing impurity by means of precipitation to the foreign protein of total salting-in protein solution except natural bioactive albumen, as preferably, described total salting-in protein solution and the volume ratio of ethanol are 1:1 ~ 3.Ethanol causes the reason of protein precipitation: be because ethanol is added to the water, solvent dielectric constant is reduced on the one hand, add the magnetism of opposite charges; Be on the other hand because ethanol is strong hydrophilicity reagent, the water of hydration on contention protein molecule surface, destroy the hydration layer of protein colloid molecular surface and molecular aggregates is precipitated, the precipitation selecting ethanol to carry out foreign protein can ensure the edible of protein isolate and application security.In general the concentration of ethanol is how many, relevant with the molecular weight of the foreign protein that can precipitate.When the volume ratio of total salting-in protein solution and ethanol is 1:1 ~ 3, in rice total salting-in protein extracting solution, the foreign protein precipitation of more than 20kDa is more and more complete, and there is bioactive 10 ~ 18kDa lower molecular weight salting-in-protein subfraction be retained in solution supernatant, purity is higher.Total salting-in protein solution can ensure different brown rice from the scope of volume ratio 1:1 ~ 3 of ethanol or/and the extraction effect of lower molecular weight biological activity protein subfraction in raw rice bran.
As preferably, the described condition left standstill is: 1 ~ 10 DEG C leaves standstill 0.5 ~ 1.5h, makes foreign protein form precipitation, is conducive to removing in follow-up process.Temperature reduces makes the reduction of the solvability of foreign protein more easily precipitate removal, is beneficial to the biological activity keeping target protein simultaneously; Time of repose is longer, and foreign protein sedimentation effect is better, leaves standstill 0.5 ~ 1.5h at 1 ~ 10 DEG C, can ensure that the foreign protein precipitation of more than 20kDa in rice is effectively removed.
As preferably, described second condition be separated is: at the centrifugal 10 ~ 30min of 7000 ~ 9000rpm, second object be separated is separated the foreign protein of more than the 20kDa of above-mentioned alcohol settling and the rice lower molecular weight biological activity protein be dissolved in supernatant (the natural bioactive albumen that namely will extract), centrifugal force and centrifugation time can affect the effect of precipitation, centrifugal rotational speed is too low or the time is too short, precipitation can not be separated completely with supernatant, cause part foreign protein to remain in supernatant, reduce target protein purity.The too high overlong time of centrifugal rotational speed, makes energy consumption increase, prolonged process, foreign protein can be made to precipitate better be separated with supernatant liquor, ensure the DNA purity of natural bioactive albumen at the centrifugal 10 ~ 30min of 5000 ~ 8000rpm.
In step 4), described aftertreatment comprises except ethanol, except salinity and drying: centrifugal vacuum method of enrichment removes ethanol, dialysis except freshen and lyophilize.Through the natural bioactive albumen that aftertreatment obtains, be lower molecular weight biological activity protein dry powder, convenient storage, transport and use.
Natural bioactive albumen prepared by the inventive method, for lower molecular weight biological activity protein dry powder, in white or yellow-white, tasteless, through SDS(sodium lauryl sulphate) to detect protein molecular weight range be 10kDa ~ 18kDa(10000 dalton ~ 18000 dalton in-polyacrylate hydrogel electrophoretic analysis), at water, ethanol, in physiological saline equal solvent, solvability is good, there is scavenging free radicals, anti-lipid peroxidation, antibacterium and fungi, suppress the biological activitys such as viral DNA synthesis, can as preparing protective foods, as foodstuff additive and being applied in makeup.
Compared with prior art, tool of the present invention has the following advantages:
The present invention is from brown rice or/and extract the method for natural bioactive albumen rice bran, adopt sodium-chlor and ethanol two kinds of food grade materials, with sodium chloride aqueous solution, the total salting-in protein of rice is extracted, obtain salting-in protein solution, adopt ethanol to carry out removing impurity by means of precipitation to the foreign protein of total salting-in protein solution except natural bioactive albumen, thus after aftertreatment, obtain the higher natural bioactive albumen of purity.The inventive method preparation cost is low, and preparation process is pollution-free, has good economic benefit and environmental benefit, is easy to industrialization scale operation.
Natural bioactive albumen prepared by the inventive method, for lower molecular weight 10kDa ~ 18kDa, dry powder, in white or yellow-white, tasteless, in water, ethanol, physiological saline equal solvent, solvability is good, there is scavenging free radicals, anti-lipid peroxidation, antibacterium and the biological activity such as fungi, the synthesis of suppression viral DNA, and can be applied in makeup as preparing protective foods, as foodstuff additive, be conducive to market-oriented large-scale promotion and utilize, have broad application prospects.
Embodiment
By following specific embodiment the present invention done and further illustrate, but should be appreciated that the present invention not limited by these contents.
Embodiment 1:
1) the organic brown rice of Great Northern Wilderness green field adopting commercially available Great Northern Wilderness green food dispensing company limited is raw material, 40 mesh sieves are crossed after pulverizing, obtain powder material, acetone is selected to carry out degreasing to powder material, powder material (g) and acetone (mL) solid-to-liquid ratio 1:3g/mL, the centrifugal 20min collecting precipitation of 6000rpm after vibration 2h, is placed in stink cupboard natural air drying and can obtains degreasing ground rice.
2) the total salting-in protein of rice extracts: take degreasing ground rice 15g and add 1.0mol/L NaCl aqueous solution 120mL, fully after mixing, and 40 DEG C of mechanical shaking extraction 2h, the centrifugal 20min of 6000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:2, mix by magnetic stirrer simultaneously, 1h is precipitated under placing 4 DEG C of conditions, the centrifugal 20min of 8000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 3 hours to distill water dialysis, obtaining natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of natural bioactive albumen is 1.5mg/g degreasing ground rice.
Embodiment 2:
1) the organic brown rice of Great Northern Wilderness green field adopting commercially available Great Northern Wilderness green food dispensing company limited is raw material, 50 mesh sieves are crossed after pulverizing, obtain powder material, acetone is selected to carry out degreasing to powder material, powder material (g) and acetone (mL) solid-to-liquid ratio 1:3g/mL, the centrifugal 20min collecting precipitation of 6000rpm after vibration 2h, is placed in stink cupboard natural air drying and can obtains degreasing ground rice;
2) the total salting-in protein of rice extracts: claim 15g degreasing ground rice to add 1.0mol/L NaCl aqueous solution 150mL, fully after mixing, and 30 DEG C of mechanical shaking extraction 2h, the centrifugal 20min of 6000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:2, mix by magnetic stirrer simultaneously, 1h is precipitated under placing 4 DEG C of conditions, the centrifugal 20min of 8000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 2 hours to distill water dialysis, obtaining natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of natural bioactive albumen is 1.8mg/g degreasing ground rice.
Embodiment 3:
1) the meticulous defatted rice bran powder adopting commercially available Tian Yu grease company limited of Jiangxi Province to produce is raw material, as degreasing ground rice;
2) the total salting-in protein of rice extracts: accurately take 15g degreasing ground rice and add 1.0mol/L NaCl aqueous solution 150mL, fully after mixing, and 30 DEG C of mechanical shaking extraction 2h, the centrifugal 20min of 6000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:2.5, mix by magnetic stirrer simultaneously, 1h is precipitated under placing 4 DEG C of conditions, the centrifugal 20min of 8000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 3 hours to distill water dialysis, obtaining natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of natural bioactive albumen is 6.2mg/g degreasing ground rice (i.e. meticulous defatted rice bran powder).
Embodiment 4:
1) the meticulous defatted rice bran powder adopting commercially available Tian Yu grease company limited of Jiangxi Province to produce is raw material, as degreasing ground rice;
2) the total salting-in protein of rice extracts: accurately take 15g degreasing ground rice and add 0.5mol/L NaCl aqueous solution 75mL, fully after mixing, and 20 DEG C of mechanical shaking extraction 4h, the centrifugal 30min of 5000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:1, mix by magnetic stirrer simultaneously, 0.5h is precipitated under placing 1 DEG C of condition, the centrifugal 30min of 7000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 3 hours to distill water dialysis, obtaining natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of natural bioactive albumen is 4.1mg/g degreasing ground rice (i.e. meticulous defatted rice bran powder).
Embodiment 5:
1) the meticulous defatted rice bran powder adopting commercially available Tian Yu grease company limited of Jiangxi Province to produce is raw material, as degreasing ground rice;
2) the total salting-in protein of rice extracts: accurately take 15g degreasing ground rice and add 0.5mol/L NaCl aqueous solution 180mL, fully after mixing, and 40 DEG C of mechanical shaking extraction 2h, the centrifugal 10min of 8000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:3, mix by magnetic stirrer simultaneously, 1.5h is precipitated under placing 10 DEG C of conditions, the centrifugal 10min of 9000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 3 hours to distill water dialysis, obtaining natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of natural bioactive albumen is 4.7mg/g degreasing ground rice (i.e. meticulous defatted rice bran powder).
Molecular weight ranges is determined:
Adopt ordinary method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method of protein molecule flow measurement, operation is with reference to the document (Jiang Shaomei of Jiang Shaomei etc., Zhu Susong, Liu family, Deng. the screening of Rice Glutelin mutant and genetic analysis. Acta Genetica Sinica, 2003,30 (7): 641-645) method listed by, carries out molecular weight analyse to the natural bioactive albumen prepared by embodiment.Wherein, not specified, percentage ratio is mass percent.
SDS-PAGE gel strength: separation gel 15%, concentrated glue 5%;
Sample preparation: get and dissolve in distilled water according to the natural bioactive albumen prepared by the embodiment of the present invention and make the solution that protein content is 0.5 μ g/ μ L, getting 100 μ L protein solutions (protein content is 50 μ g) with 25 μ L albumen loading mixed solutions [is the beta-mercaptoethanol aqueous solution of 5% by 5 μ L mass concentrations, 5 μ L mass concentrations are the tetrabromophenol sulfonphthalein aqueous solution of 0.5%, 5 μ L mass concentrations are the SDS aqueous solution of 10%, 5 μ L mass concentrations be 50% aqueous glycerin solution and 5 μ L concentration be that 250mmol/L Tris-HCl damping fluid (pH6.8) is mixed to get] mixing, boil the centrifugal 15min of 15000rpm after 5min, get 20 μ L supernatant application of samples and carry out electrophoresis on gel, simultaneously by protein standard marker solution, step 2 in embodiment) in total salting-in protein solution and step 3) solutions of biologically active proteins respectively application of sample electrophoresis, the protein content added in each sample well is 50 μ g.
Deposition condition: constant voltage 150V, electrophoresis 1.5 ~ 2 hours;
Gel-colored and decolouring: gel coomassie brilliant blue R_250 staining fluid after electrophoresis (takes in the destainer that 0.6g coomassie brilliant blue R_250 is dissolved in containing 25% dehydrated alcohol, 8% acetic acid and is made into staining fluid, use after filter paper filtering) dye 4 hours, use destainer (25% dehydrated alcohol, 8% acetic acid) to decolour again to spend the night, and observe gel electrophoresis figure.
According to its gel electrophoresis figure, by contrasting with protein standard marker, natural bioactive albumen that the embodiment of the present invention 1 ~ 5 obtains can be arrived at 13kDa ~ 16kDa, wherein, in step 2) in total salting-in protein solution in, it is except natural bioactive albumen is in addition higher than the foreign protein of 16kDa molecular weight, and step 3) solutions of biologically active proteins is exactly mainly the natural bioactive albumen of molecular weight at 13kDa ~ 16kDa.
Free-radical scavenging activity measures:
Hexichol is a kind of stabilized radical of synthetic for bitter taste phthalein free radical (DPPH), DPPH assay method is a kind of sensitive, easy, practical and measuring method of the radical scavenging activity of favorable reproducibility, is usually used in the anti-oxidant research of natural organic-compound and plant milk extract.Measuring principle: DPPH is a kind of more stable organic free radical, has strong absorption (aobvious intense violet color) near 517nm.When free-radical scavengers exists, its lone electron is paired, and absorbs and disappears or weaken, and is measured the degree absorbing and weaken, can evaluate free-radical scavengers and put forward protogenic ability by 517nm place.Its reaction pattern is:
Though this reaction is reversible reaction, when the concentration of DPPH is larger, the antioxidant AH in solution will be consumed completely.Different antioxidants is different owing to putting forward protogenic ability, can show different speed of response and free radical scavenging activity.
Measuring method: tested protein sample is diluted to 2.00,1.50,1.00,0.75,0.50,0.25, the isocyatic solution for standby of 0.10mg/mL; The DPPH ethanolic soln of another preparation 0.2mmol/L.With total sphaeroprotein for contrast, by table 1, above-mentioned reaction solution is added in tool plug test tube, shake up, after lucifuge places 30min at room temperature 25 DEG C, measure absorbancy at wavelength 517nm place.Simultaneously with V
cas positive control.The DPPH free radical scavenging activity (Scavenging Activity SA%) of tested protein sample calculates with formula (2):
In formula: A
cfor DPPH and the mixed absorbancy of dehydrated alcohol;
A
ifor DPPH and the reacted absorbancy of sample solution;
A
jfor sample solution and the mixed absorbancy of dehydrated alcohol.
Table 1 is removed DPPH and is tested application of sample table
| Absorbancy | Reaction solution |
| A c | 1.0mL DPPH+2.0mL dehydrated alcohol |
| A i | 1.0mL DPPH+2.0mL sample solution |
| A j | 1.0mL dehydrated alcohol+2.0mL sample solution |
The rice natural bioactive albumin that above-described embodiment 1 ~ 5 obtains except half-inhibition concentration (Inhibition Concentration, the IC50) concentration of DPPH free radical and free radical scavenging activity as shown in table 2.
Table 2 rice natural bioactive albumin is except the IC50 of DPPH free radical
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | |
| IC50（mg/mL） | 0.35 | 0.32 | 0.28 | 0.34 | 0.33 |
| Free radical scavenging activity | 73% | 78% | 82% | 74% | 75% |
As shown in Table 2, natural bioactive albumen prepared by the inventive method has scavenging free radicals biological activity, may be used for preparing protective foods, as foodstuff additive be applied in makeup.
Claims (3)
1., from brown rice or/and extract a method for natural bioactive albumen rice bran, it is characterized in that, comprise the following steps:
1) by brown rice or/and rice bran obtains degreasing ground rice through skimming treatment;
2) by step 1) in degreasing ground rice mix with sodium chloride aqueous solution, then through mechanical shaking extraction, the first separated and collected supernatant liquor, be total salting-in protein solution;
The concentration of described sodium chloride aqueous solution is 0.5mol/L ~ 1.5mol/L;
The quality of described degreasing ground rice and the volume ratio of sodium chloride aqueous solution are 1:5 ~ 12g/mL;
The condition of described mechanical shaking extraction was: 20 ~ 40 DEG C of mechanical shaking extractions 2 ~ 4 hours;
Described first condition be separated is: at the centrifugal 10 ~ 30min of 5000 ~ 8000rpm;
3) to step 2) in total salting-in protein solution add ethanol, mix, through leave standstill, second be separated after remove sediment fraction, obtain solutions of biologically active proteins;
Described total salting-in protein solution and the volume ratio of ethanol are 1:1 ~ 3;
The described condition left standstill is: 1 ~ 10 DEG C leaves standstill 0.5 ~ 1.5h;
Described second condition be separated is: at the centrifugal 10 ~ 30min of 7000 ~ 9000rpm;
4) by step 3) in solutions of biologically active proteins through aftertreatment except ethanol, obtain natural bioactive albumen except after salinity and drying.
2. according to claim 1 from brown rice or/and extract the method for natural bioactive albumen rice bran, it is characterized in that, step 1) in, described skimming treatment comprises: by brown rice or/and rice bran crosses 30 ~ 80 mesh sieves after pulverizing obtain powder material, in powder material, add acetone degreasing, after vibration, separation, drying, obtain degreasing ground rice.
3. according to claim 2ly it is characterized in that from brown rice or/and extract the method for natural bioactive albumen rice bran, the quality of described powder material and the volume ratio of acetone are 1:2 ~ 4g/mL.
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| CN110818780B (en) * | 2019-05-23 | 2021-08-06 | 四川农业大学 | Method for extracting component protein of low-gluten rice |
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